VIMSS, the Virtual Institute for Microbial Stress and Survival (http://vimss.lbl.gov), funded by the Dept. of Energy's Genomics:GTL Program, is dedicated to using integrated environmental data and functional genomic and comparative sequence data to understand mechanisms by which bacteria survive in uncertain environments while carrying out processes of interest for bioremediation and energy generation. To support this work, VIMSS developed a Web-based database and workbench for comparative functional genomics of bacteria and archaea. For some time, MicrobesOnline (http://www.microbesonline.org) has been providing a multi-species genome browser, operon and regulon predictions, a gene ontology browser, and a Bioinformatics Workbench for in-depth sequence analysis and community annotation of genomes. We recently incorporated data from Mikhail Gelfand's RegTransDB, which is a well-curated database of cis-regulatory sites and their regulators. VIMSS also is now releasing integrated functional genomic data with novel viewing and mining tools for microarray, proteomic, and phenotype microarray data on the website. The available data mostly are from the VIMSS Experiment/Information Data Repository (EIDR), which holds information and data on biomass production experiments and functional genomic data primarily on Desulfovibrio vulgaris and Shewanella oneidensis (and mutants of both organisms) exposed to stress conditions found at DOE field sites. Selecting an organism or gene of interest on MicrobesOnline will bring up links to information about and data viewers for VIMSS experiments conducted on that organism, or involving that gene or gene product. Information and data integration such as this serves, not only to provide insight into the regulation of stress responses of these microorganisms, but also to document VIMSS experiments, provide contextual access to experimental data of interest, and facilitate planning future experiments. We also are incorporating data released

In the absence of electron acceptors, many Desulfovibrio species grow on non-fermentable substrates via syntrophic association with hydrogen consuming methanogens. We examined the physiology of D. vulgaris Hildenborough growing syntrophically with Methanococcus maripaludis LL using a combination of transcriptional and deletion mutant analyses. Syntrophic cocultures were established in chemostats on minimal media amended with lactate but lacking electron acceptor. Replicated whole genome transcriptional analyses identified 169 and 254 genes that were significantly up- or down-regulated, respectively, relative to sulfate-limited monocultures grown at the same generation time. The majority of up-regulated genes were associated with energy production/conservation, signal transduction mechanisms, and amino acid transport/metabolism. A number of the down-regulated genes were associated with signal transduction mechanisms, inorganic ion transport/metabolism and amino acid transport and metabolism. In order to elucidate possible roles of several highly up-regulated genes associated with electron transfer and energy conservation, we constructed mutants of Desulfovibrio deleted in a subset of these genes. Cocultures developed with these mutants displayed a range of growth yields, implicating a putative carbon-monoxide induced hydrogenase (Coo, DVU2286-93) and a high-molecular weight cytochrome (Hmc, DVU0531-6) in energy conservation during syntrophic growth. Mutant monocultures grew to the same density on lactate/sulfate as the wildtype. The cooL and hmc mutants grew significantly slower and to approximately 25% yields of wildtype cocultures. Together, these data suggest a role of these genes in energy conservation of D. vulgaris Hildenborough during syntrophic growth.

Bioremediation may offer the only feasible strategy for the nearly intractable problem of metal and radionuclide contamination of soil and groundwater. To understand bioremediation in contaminated environments, it is critical to determine the organisms present in these environments, analyze their responses to stress conditions, and elucidate functional position in the environment.Methods: We used multiple molecular techniques on both sediment and groundwater to develop a better understanding of the functional capability and stress level within the microbial community in relationship to over one hundred geochemical parameters. Due to the low pH (3.5-4.5) and high contaminant levels (e.g., uranium) microbial densities and activities were low. We used a phage polymerase amplification system to construct large and small insert DNA libraries, performed metagenome sequencing, constructed clonal libraries of select functional genes (SSU rRNA gene, nirK, nirS, amoA, pmoA, and dsrAB), used a SSU rDNA Phylochip microarray (9,000 taxa), and a functional gene array (23K genes).Results: SSU rDNA analysis revealed the presence of distinct bacterial phyla, including proteobacteria, acidobacteria, and planctomycetes along the contaminant gradient. Metagenome analysis identified many of the same organisms, and diversity was lower in water than sediment. Analysis with functional gene arrays, phylochip, and specific probes for genes and organisms involved in biogeochemical cycling of C, N, and S, metal resistance, stress response, and contaminant degradation suggested that the dominant species could be biostimulated during in situ uranium reduction.Conclusion: These systems biology field studies could be enabling for strategies to attenuate metal and radionuclide contamination.

Sulfate-reducing bacteria and methanogens are found to coexist in a variety of anoxic marine sediments. In these systems they either compete for substrates or engage in successful syntrophic relationships. In our experimental setup, Desulfovibrio vulgaris Hildenborough ferments lactate, producing acetate and hydrogen. Methanococcus maripaludis, a hydrogenotrophic methanogen, then utilizes hydrogen while also incorporating limited amounts of acetate as a carbon source. Mid-log growth phase of this co-culture is achieved in 3 days growing at 37oC at which point, nearly 50percent of the initial lactate was depleted. In this study we investigate the stress response of this coculture and compare it to the D. vulgaris monoculture. Minimum Inhibitory Concentration (MIC) determinations of two environmentally relevant stressors (NO3- and NaCl) on the coculture and monoculture suggest nitrate predominantly affects M. maripaludis with a MIC of 25mM while sodium stress affects D. vulgaris with a MIC of 100mM. The response of the coculture to stressors like nitrate, nitrite, salt and peroxide was monitored by several methods. The fate of metabolites was tracked in the cultures and rates of gas evolution/utilization were measured with the Micro-Oxymax. Total biomass was measured over time with direct cell counts (including ratios of SRB: methanogen), cell protein and optical density. Metal reducing capability of log phase co-culture under NO3 stress was investigated and compared to that of under NaCl stress. Phenotype Microarray substrate utilization profiles generated by the Omnilog technology for a variety of metabolic substrates showed differential profiles for the coculture and the monoculture. Whole-genome transcriptional analysis of NaCl stressed coculture indicates up-regulation of genes coding for numerous transmembrane electron transfer enzymes.

Though considered obligate anaerobes for many years after their discovery, sulfate reducing bacteria like Desulfovibrio vulgaris Hildenborough (DvH) are found in environments with very low sulfate and in many environments that are regularly exposed to oxygen or are normally aerobic. The best growth condition for DvH, measured as increase in biomass, remains a completely anaerobic environment. However, DvH is clearly able to tolerate sub-aerobic environments and can survive exposure to air for up to 20 days. Controlled experiments were conducted to expose DvH to aerobic and microaerobic conditions (0.1% O2). Cell-wide responses were monitored via transcriptomics and proteomics measurements. Microaerobic conditions caused an overall decrease in growth without affecting the viability of the bacterium. Cellular responses to microaerobic conditions were mild and primarily included up-regulation of the putative PerR regulon, but other known oxidative stress response candidates remained unchanged. Other transcripts that show an expression profile similar to the PerR regulon genes included the cydA/B operon, encoding putative oxidative phosphorylation proteins. However, comparison with data from prior DvH functional genomics studies suggested that many of these changes could be part of a general stress response in DvH. In contrast, exposure to air produced drastic changes at both the transcriptome and proteome levels and had a detrimental effect on both growth and viability of DvH. During aerobic stress, increases in proteases and chaperones signified air exposure to be a very harsh stress in DvH. However, quantitative proteomics also indicated an accumulation of superoxide-dismutase, catalase as well as ferritins and thioredoxins, and these candidates may be critical for the survival of the small fraction of cells which survive air exposure. Our results indicated that DvH has very different responses towards microaerobic vs. aerobic exposure. Growth of DvH strains under these different O2 exposures and the data from our integrated genomics experiment are presented and have been used to improve the model for O2 stress response in DvH.

A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced at the JGI using a combination of traditional Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org, and see MicrobesOnline abstract), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Thermoanaerobacter tengcongensis), as well as other known sulfate reducers (including Archaeoglobus fulgidus and Desulfovibrio vulgaris). D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea, and offers the full complement of genes necessary for an independent lifestyle based solely on interactions with the geochemistry of the deep subsurface.

The Protein Complex Analysis Project (PCAP) has two major goals: 1. to develop an integrated set of high throughput pipelines to identify and characterize multi-protein complexes in a microbe more swiftly and comprehensively than currently possible and 2. to use these pipelines to elucidate and model the protein interaction networks regulating stress responses in Desulfovibrio vulgaris with the aim of understanding how this and similar microbes can be used in bioremediation of metal and radionuclides found in U.S. Department of Energy (DOE) contaminated sites. PCAP builds on the established research and infrastructure of another Genomics: GTL initiative conducted by the Environmental Stress Pathways Project (ESPP). ESPP has developed D. vulgaris as a model for stress responses and has used gene expression profiling to define specific sets of proteins whose expression changes after application of a stressor. Proteins, however, do not act in isolation. They participate in intricate networks of protein/protein interactions that regulate cellular metabolism. To understand and model how these identified genes affect the organism, therefore, it is essential to establish not only the other proteins that they directly contact, but the full repertoire of protein/protein interactions within the cell. In addition, there may well be genes whose activity is changed in response to stress not by regulating their expression level but by altering the protein partners that they bind, by modifying their structures, or by changing their subcellular locations. There may also be differences in the way proteins within individual cells respond to stress that are not apparent in assays that examine the average change in a population of cells. Therefore, we are extending ESPP's analysis to characterize the polypeptide composition of as many multi-protein complexes in the cell as possible and determine their stoichiometries, their quaternary structures, and their locations in planktonic cells and in individual cells within biofilms. PCAP will characterize complexes in wild type cells grown under normal conditions and also examine how these complexes are affected in cells perturbed by stress or by mutation of key stress regulatory genes. These data will all be combined with those of the ongoing work of the ESPP to understand, from a physical-chemical, control-theoretical, and evolutionary point of view, the role of multi-protein complexes in stress pathways involved in the biogeochemistry of soil microbes under a wide variety of conditions. Essential to this endeavor is the development of automated high throughput methods that are robust and allow for the comprehensive analysis of many protein complexes. Biochemical purification of endogenous complexes and identification by mass spectrometry is being coupled with in vitro and in vivo EM molecular imaging methods. Because no single method can isolate all complexes, we are developing two protein purification pipelines, one the current standard Tandem Affinity Purification approach, the other a novel tagless strategy. Specific variants ofeach of these are being developed for water soluble and membrane proteins. Our Bioinstrumentation group is developing highly parallel micro-scale protein purification and protein sample preparation platforms, and mass spectrometry data analysis is being automated to allow the throughput required. The stoichiometries of the purified complexes are being determined and the quaternary structures of complexes larger than 250 kDa are being solved by single particle EM. We are developing EM tomography approaches to examine whole cells and sectioned, stained material to detect complexes in cells and determine their localization and structures. New image analysis methods will be applied to speed determination of quaternary structures from EM data. Once key components in the interaction network are defined, to test and validate our pathway models, mutant strains not expressing these